Anti-bacterial compositions

ABSTRACT

Antibacterial compositions comprising at least one unsaturated fatty acid or a pharmaceutically acceptable salt thereof; at least one alpha-hydroxy acid or a pharmaceutically acceptable salt thereof; and at least one amino alcohol. The compositions have broad-spectrum antibacterial activity, including on pathogens displaying multi-drug resistance.

FIELD OF THE INVENTION

This invention pertains to the field of anti-bacterial compositions and,in particular, to anti-bacterial compositions comprising one or morefatty acids, one or more hydroxy acids and one or more amino alcohols.

BACKGROUND OF THE INVENTION

There is currently an urgent need for compounds and/or compositions withbroad-spectrum anti-bacterial activity. The increasing incidence ofinfectious disease caused by bacterial pathogens in both communities andhospitals is a worldwide health concern. Severe invasive infections arereported as the main complication in cancer therapies, as well as bonemarrow transplantation and major surgeries. Infection is also a majorconcern for immuno-compromised patients with haematological malignancyand/or AIDS.

Amongst bacterial pathogens, there has recently been a significantincrease of multi-drug resistance. For example, strains ofStaphylococcus aureus (methicillin-resistant or MRSA) andcoagulase-negative Staphylococci (CoNS) have become resistant to themost commonly used antibiotics, such that the only available antibioticsuniformly active against them are the glycopeptides, vancomycin andteicoplanin. S. aureus is one of the leading causes of hospital-acquiredbacteremia capable of causing a wide range of diseases ranging fromsuperficial skin infections to potentially fatal illnesses such asbloodstream infection, endocarditis and pneumonia (Diekema et al. Clin.Infect. Dis. 2001, 32:S114-132). Other human pathogens that have begunto develop resistance to multiple antibiotics include Streptococcuspneumoniae (the leading cause of nosocomial infections) and Pseudomonasaeruginosa, Haemophilus influenzae and Moraxella catarrhalis (the mostcommon community-acquired respiratory pathogens; Hoban et al. Clin.Infect. Dis. 2001, 32:S81-93).

These multidrug resistant bacteria (“superbugs”) are not restricted tohospitals alone and they can be found in diverse settings includingdaycares, schools, prisons, sports facilities, airports, healthcarefacilities, retirement homes, etc. Paper and plastics are indispensableto society and therefore need to be treated with antimicrobials toassist in the elimination of these “superbugs”.

Thus, new anti-bacterial compositions are needed to address both thegrowing resistance amongst microbes to present therapies and the generallack of efficacy of existing antibiotics against microorganisms.

In the cosmetics and food industry also there is a constant need foragents having antimicrobial properties, in particular for thepreservation of products which are otherwise perishable, but also fordirect cosmetic or therapeutic treatment of microorganisms which canhave an adverse influence on the human or animal body. Reference may bemade by way of example to microorganisms which can cause body odour,acne, mycoses or the like.

The antimicrobial properties of free fatty acids have been known formany years (Kabara J. et al. Antimicrobial Agents and Chemotherapy, July1972; 2(1): pp 23-28). Bergson et al. (Antimicrobial Agents andChemotherapy, November 2001, pp 3209-3212), reported that both capricand lauric acid were effective in killing the yeast Candida albicans.Sun et al. (Chemico-Biological Interactions 140 (2002), pp 185-198),identified the superior microbicidal properties of caprylic, capric andlauric acid, concluding that lauric was most potent against grampositive bacteria while caprylic was optimal against gram negativeorganisms.

WO 2011/061237 discloses antimicrobial compositions comprising freefatty acids emulsified with membrane lipids or hydrolysed derivativesthereof, and pharmaceutical formulations comprising same. Thecompositions can be used in the treatment or prophylaxis of microbialinfections. They can also regulate the rate of blood clotting renderingthem suitable for incorporation in catheter locking solutions and foruse in wound care.

WO 99/51218 discloses a biocidal composition of a blend of acidssubstantially free of benzoic acid or a derivative thereof andcomprising a mixture of lactic acid and at least one other acid selectedfrom formic acid, acetic acid and propionic acid. In GB 1,194,863preservative composition for crops is described comprising 70 wt. %phosphoric acid, 20 wt. % propionic acid and 5 wt. % lactic acid.

U.S. Pat. No. 7,727,568 discloses an antimicrobial compositioncomprising a mixture of at least 20 wt. % lactic acid or a derivativethereof and an inorganic acid selected from a nitrogen, sulfur, andphosphorous acid, and mixtures thereof for use in animal nutrition. Thecomposition can further comprise at least one other acid selected fromacetic acid, fumaric acid, gluconic acid, (iso)butyric acid, sorbicacid, (iso)valeric acid, maleic acid, malic acid, capronic acid, benzoicacid, and citric acid.

WO2014/035246 discloses antimicrobial compositions comprising at leastone free fatty acid or a derivative and/or a pharmaceutically acceptablesalt thereof, at least one carboxylic acid or a pharmaceuticallyacceptable salt thereof; and/or at least one carbohydrate or apharmaceutically acceptable salt thereof, wherein the carbohydrate isselected from a hydrogenated carbohydrate, a monosaccharide, adisaccharide, a polysaccharide and combinations thereof. Thecompositions of this reference are aimed to provide an antimicrobialcomposition for treating or preventing the first stage in pathogenesisin order to prevent infections. This reference also discloses that acomposition comprising a combination of at least one free fatty acid andat least one carboxylic acid exhibit disinfecting properties, whereasthe combination of at least one free fatty acid and at least onecarbohydrate, optionally in combination with at least one carboxylicacid, exerts a dual antimicrobial effect.

WO2009/140062 discloses use of amino alcohols as additives forhydrocarbonaceous compositions, such as petroleum and fuels, to improvethe corrosion and microbial resistance of hydrocarbonaceouscompositions. This reference also discloses that the particularlypreferred amino alcohols for the desired result are2-amino-2-methyl-1-hexanol, 2-amino-2-ethyl-1-pentanol,2-amino-2-methyl-1-octanol, 2-amino-2-ethyl-1-heptanol,2-amino-2-propyl-1-hexanol, (1-aminocyclohexyl)methanol,(1-aminocyclooctyl)methanol, 2-amino-2-phenyl-1-propanol,(1-aminocyclopentyl)methanol, and mixtures thereof.

There remains a need for antimicrobial compounds and/or compositionsthat have improved and/or broad spectrum anti-bacterial activity.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel compositionhaving improved anti-bacterial activity. In accordance with an aspect ofthe present invention, there is provided an antibacterial compositioncomprising at least one unsaturated fatty acid or a pharmaceuticallyacceptable salt thereof, wherein the free fatty acid is selected from afree fatty acid having from 6 to 16 carbon atoms; at least onealpha-hydroxy acid or a pharmaceutically acceptable salt thereof; and atleast one amino alcohol.

In accordance with another aspect of the present invention, there isprovided a pharmaceutical formulation comprising a composition asdefined above, and a pharmaceutically acceptable carrier.

In accordance with another aspect of the present invention, there isprovided a use of the pharmaceutical formulation of the presentinvention for inhibiting growth and/or proliferation of a microbe.

In accordance with another aspect of the present invention, there isprovided a method of killing and/or inhibiting the growth of microbes ona substrate comprising applying an effective amount of the antibacterialcomposition as defined above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel anti-bacterial compositions anduses thereof. In the context of the present invention, the term“anti-bacterial” refers to the inhibition, prevention or eradication ofthe growth or proliferation of bacteria and to the inhibition,prevention or eradication of the growth or proliferation of bacterialcells.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

The term “alkyl” refers to a straight chain or branched, alkyl group ofone to ten carbon atoms. This term is further exemplified by such groupsas methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, 1-butyl (or2-methylpropyl), and the like.

The term “amino” refers to the group NRR′, where R and R′ mayindependently be hydrogen, lower alkyl, or substituted alkyl.

The terms “therapy” and “treatment,” as used interchangeably herein,refer to an intervention performed with the intention of alleviating thesymptoms associated with, preventing the development of, or altering thepathology of a disease, disorder or condition. Thus, the terms therapyand treatment are used in the broadest sense, and include the prevention(prophylaxis), moderation, management, reduction, or curing of adisease, disorder or condition at various stages. Prevention orreduction of the progression of a disease, disorder or condition isencompassed by these terms. Also encompassed by these terms is anintervention resulting in an alteration of physiology and/orbiochemistry of a living subject. Those in need of therapy/treatmentinclude those already having the disease, disorder or condition as wellas those prone to, or at risk of developing, the disease, disorder orcondition and those in whom the disease, disorder or condition is to beprevented. The therapeutic application of compounds of the invention,therefore, refers to a therapy or treatment, as defined herein.

The terms “subject” or “patient,” as used herein, refer to an animal inneed of treatment, including humans and other mammals.

Administration of the composition of the present invention “incombination with” one or more further therapeutic agents, is intended toinclude simultaneous (concurrent) administration and consecutiveadministration. Consecutive administration is intended to encompassvarious orders of administration of the therapeutic agent(s) and thecompound(s) to the subject.

The term “inhibit,” as used herein, means to reduce, halt or hold incheck, and thus inhibition may be complete or partial and may be ofshort or long term duration. The term may be used in the context ofinhibiting a process or action already begun or it may be used in thecontext of inhibiting initiation of a process or action.

As used herein, the term “about” refers to approximately a +/−10%variation from a given value. It is to be understood that such avariation is always included in any given value provided herein, whetheror not it is specifically referred to.

The Compositions

The present invention provides an antibacterial composition comprisingat least one unsaturated fatty acid or a pharmaceutically acceptablesalt thereof; at least one alpha-hydroxy carboxylic acid or apharmaceutically acceptable salt thereof; and at least one aminoalcohol.

The unsaturated fatty acids of the present invention have from 6 to 16carbon atoms, preferably the free fatty acid has from 8 to 12 carbonatoms. In one embodiment, the free fatty acid is undecylenic acid.

The alpha hydroxy acids of the present invention can be selected fromglycolic acid, lactic acid, citric acid, mandelic acid, oxalic acid, andmalonic acid. In one embodiment, the alpha hydroxy acid is lactic acid.

The amino alcohol of the present invention can have a formula:

wherein R¹ and R³ are each independently H, linear or branched alkyl, R²and R⁴ are each independently H, linear or branched alkyl; and R5 isabsent or is a C₁-C₆ alkylene.

In one embodiment, in the formula (I) above, R5 is absent, R2 and R4 areboth C1-C6 alkyl. In one embodiment, the amino alcohol is2-amino-2-methyl-1-propanol.

In one embodiment, in the formula (I) above, R5 is absent, R2 is C1-C6alkyl and R4 is CH₂OH.

In one embodiment, the amino alcohol is amino methyl propane diol(AMPD).

In one embodiment, the amino alcohol is monoethanolamine (MEA).

The individual concentrations of unsaturated fatty acid, alpha hydroxyacid and amino alcohol can be in the range of about 5% to about 90% byweight of the total weight of the composition.

In one embodiment the concentration of the unsaturated fatty acid isabout 5%, 10%, 15%, 20%, 25%, 30%, 235%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or a percentage between any two of these values.

In one embodiment the concentration of the alpha hydroxy acid is about5%, 10%, 15%, 20%, 25%, 30%, 235%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or a percentage between any two of these values.

In one embodiment the concentration of the amino alcohol is about 5%,10%, 15%, 20%, 25%, 30%, 235%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90% or a percentage between any two of these values.

In one embodiment, in the composition of the present invention theamount of alpha hydroxy acid is in the range of about 10% to about 40%by weight of the total weight of the composition, the amount ofunsaturated fatty acid is in the range of about 40% to about 80% byweight of the total weight of the composition, and the amount of aminoalcohol is in the range of about 5% to about 25% by weight of the totalweight of the composition. In one embodiment, in the composition of thepresent invention the amount of alpha hydroxy acid is in the range ofabout 20% to about 30% by weight of the total weight of the composition,the amount of unsaturated fatty acid is in the range of about 50% toabout 70% by weight of the total weight of the composition, and theamount of amino alcohol is in the range of about 10% to about 15% byweight of the total weight of the composition. In one embodiment, in thecomposition of the present invention the amount of alpha hydroxy acid isin the range of about 10% to about 15% by weight of the total weight ofthe composition, the amount of unsaturated fatty acid is in the range ofabout 50% to about 70% by weight of the total weight of the composition,and the amount of amino alcohol is in the range of about 20% to about30% by weight of the total weight of the composition

In one embodiment, the composition of the present invention comprisesabout 28.00 wt. % of alpha hydroxy acid, about unsaturated fatty acid58.00 wt. % and about 14.00 wt. % of amino alcohol (95%). In oneembodiment, the composition of the present invention comprises about25.00 wt. % of alpha hydroxy acid, about unsaturated fatty acid 55.00wt. % and about 20.00 wt. % of amino alcohol (95%). In one embodiment,the composition of the present invention comprises about 30.00 wt. % ofalpha hydroxy acid, about unsaturated fatty acid 60.00 wt. % and about10.00 wt. % of amino alcohol (95%).

The antibacterial composition may further comprise at least oneviscosity-enhancing agent, i.e. thickening agent. Preferably theviscosity-enhancing agent is selected from xanthan gum, alginic acid,agar, carrageenan, locust bean gum, pectin, cellulose derivatives,gelatin and combinations thereof.

The antibacterial composition may comprise at least one emulsifyingagent, such as polysorbate (Tween) 20, polysorbate 40, polysorbate 60,polysorbate 80, polyoxyethylene glycol alkyl ethers, glucoside alkylethers, polyoxyethylene glycol octylphenol ethers, polyoxyethyleneglycol alkylphenol ethers, glycerol alkyl esters, poloxamers, polyoxylcastor oil, and combinations thereof. More preferably the emulsifyingagent is polysorbate 80.

Uses of the Anti-Bacterial Compositions

The present invention provides for the use of the compositions disclosedherein for the inhibition, prevention or eradication of the growthand/or proliferation of bacteria, either alone or in combination withknown anti-microbial agents.

In one embodiment, the present invention provides a method of inhibitingbacterial growth by contacting a bacterium with an effective amount of acomposition as disclosed herein.

The compositions have broad spectrum anti-bacterial activity, in whichcase they may be used against gram-positive and/or gram-negativebacteria.

Examples of gram-positive bacteria include, Clostridium difficile,Clostridium perfringens-vegetative cells, Clostridiumsporogenes-vegetative cells, Enterococcus faecalis-vancomicyn resistant(VRE), Enterococcus faecium, Micrococcus luteus, Mycobacteriumsmegmatis, Staphylococcus aureus, Staphylococcus aureus-methicillinresistant (MRSA), Staphylococcus aureus-vancomicyn resistant (VRSA),Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pneumonia, Streptococcus pneumoniae-Drug Resistant,Streptococcus pyogenes, Streptococcus-Group A-Erythromycin-resistant,Streptococcus-Group B-Clindamycin-resistant and Pseudomonas aeruginosa,Streptococcus pyogenes and/or Streptococcus faecalis.

Examples of gram-negative bacteria include Acinetobacter baumannii,Acinetobacter—multi drug resistant, Bacteroides fragilis, Bordetellapertussis, Burkholderia cepacia, Camplylobacter species,Camplylobacter—drug resistant, Enterobacter aerogenes,Enterobacteriaceae (ESBLs), Enterobacteriaceae—carbapenem-resistant,Escherichia coli (cfu/g) 4.4×10⁵, Escherichia coli (E.coli)-(Resistant), Haemophilus influenza, Helicobacter pylori,Klebsiella oxytoca, Klebsiella pneumoniae pneumoniae (CRE), Neisseriagonorrhoeae, Neisseria gonorrhoeae-drug resistant, Neisseriameningitides, Proteus mirabilis, Pseudomonas aeruginosa (cfu/g) 3.5×10⁵,Pseudomonas aeruginosa (Regular), Pseudomonas aeruginosa—multi drugresistant, Salmonella-non-typhoidal-drug resistant, Salmonella typhi,Salmonella typhi-drug resistant, Serratia marcescens, Shigella sonnei,Shigella-drug-resistant, Vibrio cholerae, Enterobacter, and/orKlebsiella pneumonia.

In one embodiment, examples of bacteria that may be inhibited by thecompositions of the present invention include, but are not limited to,Enterobacter faecalis, Enterococcus faecium, Escherichia coli,Escherichia coli O157:H7, Escherichia coli (cfu/g) 4.4×10⁵,Staphylococcus aureus, Staphylococcus aureus K147, Staphylococcusepidermidis, Pseudomonas aeruginosa and Klebsiella pneumonia.

It is well-established in the field of microbiology that manymultidrug-resistant strains of bacteria have emerged in the recent pastand will continue to emerge with the continued use of standardantibiotics. Examples of currently known resistant strains of bacteriainclude methicillin-resistant Staphylococcus aureus (MRSA), andvancomycin-resistant Enterococcus faecium.

In one embodiment, the compositions of the present invention can be usedto inhibit growth of such multidrug-resistant strains. In oneembodiment, the compositions of the present invention are used toinhibit the growth of MRSA and/or Enterococcus faecium.

In one embodiment, the compositions of the present invention are used inthe preparation of antibiotic compositions.

The compositions of the present invention can be used as the activeingredient in anti-bacterial cleansers, polishes, paints, sprays, soaps,or detergents. In such cases, antibacterial composition of the presentinvention can generally be used in quantities of between about 0.1% andabout 20% by weight of the final product. In one embodiment, the amountof antibacterial composition is about 0.1% to 5% by weight. In oneembodiment the amount of antibacterial composition is about 0.3% toabout 5% by weight.

These compositions can also be included as an anti-bacterial agent incosmetic, personal care, household and industrial products, for example,to improve shelf-life by inhibiting the growth of microbes within theproducts.

The compositions may be formulated for application to surfaces toinhibit the growth of a bacterial species thereon, for example, surfacessuch as countertops, desks, chairs, laboratory benches, tables, floors,sinks, showers, toilets, bathtubs, bed stands, tools or equipment,doorknobs and windows. Alternatively, the compositions may be formulatedfor laundry applications, for example, for washing clothes, towels,sheets and other bed linen, washcloths or other cleaning articles.

The antibacterial cleansers, polishes, paints, sprays, soaps, ordetergents according to the present invention can optionally containsuitable solvent(s), carrier(s), thickeners, pigments, fragrances,deodorisers, emulsifiers, surfactants, wetting agents, waxes, or oils.The cleansers, polishes, paints, sprays, soaps, and detergents accordingto the present invention are useful in institutions, such as in hospitalsettings for the prevention of nosocomial infections, as well as in homesettings.

In addition, the invention contemplates the use of the compositions informulations to kill or inhibit the growth of bacterial species in foodpreparations, or to sterilise surgical and other medical equipment andimplantable devices, including prosthetic joints. The compositions canalso be formulated for use in the in situ sterilisation of indwellinginvasive devices such as intravenous lines and catheters, which areoften foci of infection.

The present invention further contemplates the use of these compositionsas the active ingredient in personal care items, such as soaps,deodorants, shampoos, mouthwashes, toothpastes, and the like. Manycompositions used in personal care applications are susceptible tobacterial growth and it is thus desirable to incorporate into thesecompositions an effective anti-bacterial material.

In one embodiment, the present invention provides a formulationcontaining a composition as defined herein for external use as apharmaceutically acceptable skin cleanser. In one embodiment, thecompositions of the present invention can also be used as dermocosmeticcompositions.

The anti-bacterial agent may be incorporated into the personal careformulation using techniques known in the art. Thus, the anti-bacterialagent may be added to the personal care formulation as a solution,emulsion or dispersion in a suitable liquid medium. Alternatively, theanti-bacterial agent may be added, undiluted, to the personal careformulation or may be added with a solid carrier or diluent. Theanti-bacterial agent may be added to the pre-formed personal careformulation or may be added during the formation of the personal careformulation, either separately or premixed with one of the othercomponents of the formulation.

The antibacterial composition of the present invention can generally beused in quantities of between 0.1% and 20% by weight of the personalcare compositions. In one embodiment, the amount of antibacterialcomposition is between 0.1% and 5% by weight. In one embodiment theamount of antibacterial composition is about 0.3% to about 5% by weight.

Pharmaceutical Formulations and Administration of Anti-BacterialCompositions

For use as therapeutic agents in the treatment of bacterial infections,or disorders or diseases associated therewith in a subject, theanti-bacterial compositions of the present invention are typicallyformulated prior to administration. Therefore, the present inventionprovides pharmaceutical formulations comprising one or more compositionsof the present invention and a pharmaceutically-acceptable carrier,diluent, or excipient. The present pharmaceutical formulations areprepared by standard procedures using well-known and readily availableingredients. In making the compositions of the present invention, theactive ingredient will usually be mixed with a carrier, or diluted by acarrier, or enclosed within a carrier, and may be in the form of acapsule, sachet, paper, or other container.

The pharmaceutical formulations comprising the anti-bacterialcompositions according to the present invention may be formulated in anumber of ways depending upon the desired treatment and upon the area tobe treated. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary, e.g.by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g. intrathecal or intraventricular,administration.

For administration to an individual for the treatment of an infection ordisease, the present invention also contemplates the formulation of thepharmaceutical formulations comprising the anti-bacterial compositioninto oral dosage forms such as tablets, capsules and the like. For thispurpose, the composition can be combined with conventional carriers,such as magnesium carbonate, magnesium stearate, talc, sugar, lactose,pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, sodiumcarboxymethyl-cellulose, low melting wax, cocoa butter and the like.Diluents, flavouring agents, solubilizers, lubricants, suspendingagents, binders, tablet-disintegrating agents and the like can also beemployed, if required. The anti-microbial compositions can beencapsulated with or without other carriers. In accordance with thepresent invention, the proportion of anti-bacterial composition(s) inany solid and liquid formulation will be at least sufficient to impartthe desired activity to the individual being treated upon oraladministration. The present invention further contemplates parenteralinjection of the anti-bacterial compositions, in which case thecompositions are formulated as a sterile solution containing othersolutes, for example, enough saline or glucose to make the solutionisotonic.

For administration by inhalation or insufflation, the anti-microbialcompositions can be formulated into an aqueous or partially aqueoussolution, which can then be utilized in the form of an aerosol. Aqueousformulations of the anti-bacterial compositions of the present inventionmay also be used in the form of ear or eye drops, or ophthalmicsolutions. The present invention further contemplates topical use of theanti-bacterial compositions. For this purpose they can be formulated asdusting powders, creams or lotions in pharmaceutically acceptablevehicles, which are applied to affected portions of the skin.

Compositions intended for oral use may be prepared according toprocedures known in the art for the manufacture of pharmaceuticalformulations and such formulations may further contain one or moresweetening agents, flavouring agents, colouring agents, preservingagents, or a combination thereof, in order to provide pharmaceuticallyelegant and palatable preparations. Tablets typically contain theanti-bacterial composition(s) in admixture with non-toxicpharmaceutically acceptable excipients suitable for the manufacture oftablets, such as inert diluents, for example, calcium carbonate, sodiumcarbonate, lactose, calcium phosphate or sodium phosphate; granulatingand disintegrating agents, for example, corn starch, or alginic acid;binding agents, for example, starch, gelatine or acacia, and lubricatingagents, for example, magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

Formulations for oral use may also be presented as hard gelatinecapsules wherein the anti-bacterial composition(s) is mixed with aninert solid diluent, for example, calcium carbonate, calcium phosphateor kaolin, or as soft gelatine capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

Aqueous suspensions typically contain the anti-bacterial composition(s)in admixture with excipients suitable for the manufacture of aqueoussuspensions, such as suspending agents (for example, sodiumcarboxylmethylcellulose, methyl cellulose, hydropropylmethyl cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);dispersing or wetting agents such as a naturally-occurring phosphatide(for example, lecithin), or condensation products of an alkylene oxidewith fatty acids (for example, polyoxyethylene stearate), orcondensation products of ethylene oxide with long chain aliphaticalcohols (for example, hepta-decaethyleneoxycetanol), or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand a hexitol (for example, polyoxyethylene sorbitol monooleate), orcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides (for example, polyethylene sorbitanmonooleate). The aqueous suspensions may further contain one or morepreservatives, for example, ethyl, or n-propyl-p-hydroxy benzoate; oneor more colouring agents; one or more flavouring agents, or one or moresweetening agents, such as sucrose or saccharin, or a combinationthereof.

Oily suspensions may be formulated by suspending the anti-bacterialcomposition(s) in a vegetable oil, for example, peanut oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.The oily suspensions may contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents such as thoseset forth above, and flavouring agents may be added to provide palatableoral preparations. These compositions may be preserved by the additionof an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the anti-bacterialcomposition in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those mentioned above.Additional excipients, for example, sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical formulations of the present invention may also be in theform of oil-in-water emulsions. The oil phase may be a vegetable oil,for example, olive oil or peanut oil, or a mineral oil, for example,liquid paraffin, or mixtures thereof. Suitable emulsifying agents may benaturally-occurring gums (for example, gum acacia or gum tragacanth);naturally-occurring phosphatides (for example, soy bean lecithin), andesters or partial esters derived from fatty acids and hexitol anhydrides(for example, sorbitan monooleate), and condensation products of thepartial esters with ethylene oxide (for example, polyoxyethylenesorbitan monooleate). The emulsions may also contain sweetening andflavouring agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain one or more demulcents, preservatives orflavouring and colouring agents, or combinations thereof.

The pharmaceutical formulations may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to known art using suitable dispersing or wettingagents and suspending agents as described above. The sterile injectablepreparation may also be a solution or a suspension in a non-toxic,parentally acceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. Typically, a bland fixed oil isemployed for this purpose such as a synthetic mono- or diglyceride. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectables. Adjuvants, such as local anaesthetics, preservatives andbuffering agents, may also be included in the injectable formulation.

The composition(s) of the present invention may be administered,together or separately, in the form of suppositories for rectal orvaginal administration of the composition. These compositions can beprepared by mixing the composition with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal/vaginal temperature and will therefore melt to release thecomposition. Examples of such materials include cocoa butter andpolyethylene glycols.

Another formulation of the present invention employs transdermaldelivery devices (“patches”). Such transdermal patches may be used toprovide continuous or discontinuous administration/application of theanti-bacterial compositions of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art (see, forexample, U.S. Pat. No. 5,023,252; issued Jun. 11, 1991, incorporatedherein by reference in its entirety). Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

In one embodiment, the composition(s) of the present invention can beincorporated into medical dressings such as Tegaderm pad from 3M (whichact as traditional sponge gauze, a bacterial barrier just helping toreduce the risk of infection). In Tegaderm structure, the mainbiocompatible wound dressing part is made of cellulose paper fibrecoated with silicone material, and all supporting and adhering parts aremade of synthetic materials such as: polyethylene, polyurethane,polyester and acrylate polymer.

It may be desirable or necessary to introduce the pharmaceuticalformulations to the brain, either directly or indirectly. Directtechniques usually involve placement of a drug delivery catheter intothe host's ventricular system to bypass the blood-brain barrier. Anexample of such an implantable delivery system, used for the transportof biological factors to specific anatomical regions of the body, isdescribed in U.S. Pat. No. 5,011,472, incorporated herein by referencein its entirety.

The dosage of the anti-bacterial composition to be administered is notsubject to defined limits, but will usually be an effective amount. Ingeneral, the dosage will be the equivalent, on a molar basis, of thepharmacologically active free form produced from a dosage formulationupon the metabolic release of the active free drug to achieve itsdesired pharmacological and physiological effects. The pharmaceuticalcompositions are typically formulated in a unit dosage form, each dosagecontaining from, for example, about 0.05 to about 100 mg of theanti-bacterial composition. The term “unit dosage form” refers tophysically discrete units suitable as unitary dosages for administrationto human subjects and other animals, each unit containing apredetermined quantity of anti-bacterial composition calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient.

Typical daily dosages of the anti-bacterial compositions fall within therange of about 0.01 to about 200 mg/kg of body weight in single ordivided dose. However, it will be understood that the amount of thecomposition actually administered will be determined by a physician, inthe light of the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual compositionadministered, the age, weight, and response of the individual patient,and the severity of the patient's symptoms, and therefore the abovedosage ranges are not intended to limit the scope of the invention inany way. In some instances dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful side effect,for example, by first dividing larger doses into several smaller dosesfor administration throughout the day.

Compositions of the present invention formulated for topicaladministration are suitable in the treatment and/or prevention ofbacterial infections of the skin and mucosa.

Topical pharmaceutical and/or dermocosmetic formulations according tothe invention comprise the antibacterial composition mixed with one ormore suitable excipients and may be, for example, in the form of cream,ointment, gel, gum, toothpaste, mouthwash or shampoo.

The pharmaceutical formulations and/or the dermocosmetic formulationscan comprise about 0.1% to about 20% by weight of the antibacterialcomposition of the present invention. In one embodiment, the amount ofantibacterial composition is about 0.1% to about 10% by weight. In oneembodiment, the amount of antibacterial composition is about 0.1% toabout 5% by weight. In one embodiment the amount of antibacterialcomposition is about 0.3% to about 2% by weight.

Examples of suitable excipients that may be used in the compositionsaccording to the invention are solvents, diluents, gliding agents,preservatives, gums, sweeteners, coating agents, binders, disintegratingagents, lubricants, suspending agents, dispersing agents, colorants,flavouring agents, non-stick agents, surfactants, plasticisers,emulsifiers, chelating agents and emollients.

The solvent preferably used is water, but alcohols or other organicsolvents may also be used, possibly mixed with water.

The choice of excipients is part of the normal knowledge of one skilledin the art, and will mainly depend on the pharmaceutical and/ordermocosmetic form chosen.

For example, a cream can be prepared by incorporating the anti-bacterialcomposition of the present invention in a topical carrier consisting ofliquid paraffin, dispersed in an aqueous medium by means of lubricants.An ointment can be prepared by mixing TSP with a topical carrier such asmineral oil or wax. A gel can be prepared by mixing TSP with a topicalcarrier containing a gelling agent.

The pharmaceutical and/or dermocosmetic composition according to theinvention may also be a, woven or non-woven, material coated and/orimpregnated with a mixture of the anti-bacterial composition with asuitable carrier or a matrix in which the anti-bacterial composition isdispersed so that it comes into contact with the skin for transdermaladministration. Specific examples are bandage, gauze, towelettes, etc.

The choice of type of pharmaceutical and/or dermocosmetic form willdepend mainly on the area to be treated and is part of the normalknowledge of one skilled in the art. For example, a gum or mouthwash maybe more suitable to treat the oral cavity, whereas a cream, ointment,lotion or towelettes may be suitable for the skin of the face.

The term “skin” is used according to the present in its conventionalmeaning, namely an external organ including the epithelial tissue. Theterm “mucosa” is also used with its usual meaning, which relates to allthe mucosal barriers in the body, such as the gastrointestinal,pulmonary, sublingual, buccal, rectal, vaginal, nasal, urethral andocular barriers.

The compositions according to the invention are preferably applied bytopical administration directly to the area of the skin or mucosa whichpresents, or is assumed to present, a bacterial infection or otherdisorders caused by the presence of microbes. The infection oftenoriginates in a part of the skin or mucosa which presents a lesion, suchas a wound, laceration or burn. In such case, the composition accordingto the invention can be applied directly to the lesion and/or thesurrounding area.

The compositions of the present invention can also be used in thetreatment and/or prevention of several disorders of the skin and mucosa,which are known to be caused by bacteria, for example, psoriasis,eczema, acne, etc. Other treatments may include wound care, and burncare, etc.

Anti-Bacterial Activity of Compositions

The anti-bacterial activity of a candidate composition can be testedusing standard techniques known in the art. As is known in the art,anti-bacterial activity of a composition or composition may result inthe killing of bacterial cells (i.e. bacteriocidal activity), or it mayresult in the slowing or arrest of the growth of bacterial cells (i.e.bacteriostatic activity). Thus the compositions of the present may bebacteriocidal and/or bacteriostatic. Compositions of the presentinvention that slow or arrest bacterial cell growth may be useful incombination treatments with other known anti-bacterial agents.

In Vitro Testing

In vitro methods of determining the ability of candidate compositions toinhibit, prevent or eradicate the growth of bacterial cells arewell-known in the art. In general, these methods involve contacting aculture of the cells of interest with various concentrations of thecandidate composition and monitoring the growth of the cell culturerelative to an untreated control culture. A second control culturecomprising cells contacted with a known anti-bacterial agent may also beincluded in such tests, if desired.

Anti-bacterial effects can be expressed as the percentage (%) inhibitionof growth of a given micro-organism over a pre-determined period of timeby treatment with a single concentration of a candidate composition.This method provides a rapid method of assessing the ability of acomposition to inhibit bacterial growth, for example, prior toconducting more in-depth tests, such as MIC determinations or in vivotesting. An example of such an testing is in-vitro Time-Kill Methodwhich is well known in the art.

Toxicity Testing

It is important that the anti-bacterial compositions of the presentinvention exhibit low toxicity.

In vitro acute toxicity testing of a composition of the presentinvention can be performed using mammalian cell lines (see, for example,Ekwall, B., Ann. N.Y. Acad. Sci., (1983) 407:64-77). Selection of anappropriate cell line is dependent on the potential application of thecandidate composition and can be readily determined by one skilled inthe art.

In vivo toxicity testing can be performed by standard methodology, forexample, by injecting or introducing varying concentrations of thecandidate composition into an appropriate animal model. The compositioncan be injected once, or administration can be repeated over severaldays. The toxic effects of the composition can be evaluated over anappropriate time period by monitoring the general health and body weightof the animals.

After the completion of the period of assessment, the animals can besacrificed and the appearance and weight of the relevant organsdetermined.

In Vivo Testing

The ability of a test composition to act as an anti-bacterial agent canalso be tested in vivo using standard techniques. A number of animalmodels are known in the art that are suitable for testing the activityof anti-bacterial compositions and are readily available.

Methods for conducting in vivo tests to determine the activity ofanti-bacterial compositions are well-known in the art. Typically, invivo testing comprises introducing a selected micro-organism into theappropriate animal model in a sufficient amount to cause infection,followed by administration of one or more doses of the test composition.Methods of administration will vary depending on the composition beingemployed, but can be, for example, by way of bolus infusion into asuitable vein (such as the tail vein of mice or rats), or by oraladministration. Animals treated with a known anti-bacterial agent and/orwith a saline or buffer control solution serve as controls. Repeat dosesof the test composition may be administered to the animal, if necessary,at appropriate time intervals. The animals are subsequently monitoreddaily for mortality.

Additional Tests

In addition to the above tests, the compositions of the invention can besubmitted to other standard tests, such as stability tests,bioavailability tests and the like. As will be readily apparent to oneskilled in the art, compositions in accordance with the presentinvention will need to meet certain criteria in order to be suitable forhuman use and to meet regulatory requirements. Thus, once a compositionof the invention has been found to be suitable for animaladministration, standard in vitro and in vivo tests can be conducted todetermine information about the metabolism and pharmacokinetic (PK) ofthe compositions and combinations (including data on drug-druginteractions where appropriate) which can be used to design humanclinical trials.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It should be understood that theseexamples are for illustrative purposes only. Therefore they should notlimit the scope of the invention in any way.

EXAMPLES

An exemplary composition of the present invention (composition A) wasprepared by mixing 50 grams of lactic acid, 105 grams of undecylenicacid and 25 grams of AMP-95%.

Composition A is soluble in most solvents (i.e., acetone, chloroform,methanol, ethanol, benzene, DMAC, DMSO) with no loss of bioactivity andcan be solubilized in aqueous medium using a co-solvent system. Physicalproperties of the composition remained the same after heating (no colorchange or gelation or evaporation was observed).

Example 1 In Vitro Inhibition of Gram Positive Bacteria

The anti-bacterial effect of the exemplary composition A was evaluatedat two different concentrations versus suspensions of Enterococcusfaecalis VRE (ATCC #51575) and Staphylococcus aureus aureus MRSA (ATCC#33591).

Testing was conducted in accordance with a Non-GLP evaluation of onetest material for its antibacterial properties when challenged with twomicroorganism species using an in-vitro Time-Kill Method. The testmaterial was prepared at two different concentrations prior toevaluation. Test Solution #1 was prepared by diluting the test materialin the ratio of 1.0 mL of concentrated test material to 100 mL ofsterile Water-for-Irrigation, USP (WFI) (1:100 [v/v] dilution). TestSolution #2 was prepared by diluting the test material in the ratio of0.1 mL of Composition A to 100 mL of WFI (1:1,000 [v/v] dilution). A 0.1mL aliquot of a challenge suspension was inoculated into a test tubecontaining 9.9 mL of a Test Solution and mixed thoroughly using a vortexmixer. Each challenge suspension was exposed to each Test Solution for10 minutes, timed using a calibrated minute/second timer. After theexposure time had elapsed, a 1.0 mL aliquot was transferred from thetube containing Test Solution/inoculum into a separate sterile test tubecontaining 9.0 mL of Butterfield's Phosphate Buffer solution withproduct neutralizers (BBP++), and mixed thoroughly using a vortex mixer.Ten-fold dilutions were prepared in neutralizing solution, mixingthoroughly using a vortex mixer between dilutions. 1.0 mL and/or 0.1 mLaliquots of each dilution were pour-plated, in duplicate, using TrypticSoy Agar with product neutralizers (TSA+).

Tables 1 and 2 present the initial population (CFU/mL) and post-exposurepopulations (CFU/mL) of each challenge species, and the Log₁₀ andpercent reductions produced by each Test Solution following a 10-minuteexposure.

TABLE 1 Test Solution #1 - Composition A 1:100 [v/v] dilution¹ Post-Microorganism Species Inoculum Level Exposure Exposure Log10 Percent(ATCC #) (CFU/mL) Time Population Reduction Reduction Enterococcusfaecalis  3.60 × 10⁷ 10 minutes <1.00 × 10¹ 6.5563 99.9999% VRE (ATCC#51575) Staphylococcus aureus 1.1150 × 10⁷ 10 minutes  1.50 × 10¹ 5.871299.9999% aureus MRSA (ATCC #33591)

TABLE 2 Test Solution #2 - Composition A 1:1,000 [v/v] dilution¹Inoculum Post-Exposure Microorganism Species Level Exposure PopulationLog10 Percent Enterococcus faecalis  3.60 × 10⁷ 10 minutes <1.00 × 10¹6.5563 99.9999% VRE (ATCC #51575) Staphylococcus aureus 1.1150 × 10⁷ 10minutes <1.00 × 10¹ 6.0473 99.9999% aureus MRSA (ATCC #33591)

Notes:

-   -   1. Concentrated Test Composition was diluted with sterile        Water-for-Irrigation, USP, prior to evaluation.

Example 2 In vitro Inhibition of Gram Negative Bacteria

The anti-bacterial effect of the test material, Composition A, wasevaluated at two different concentrations versus suspensions ofEscherichia coli (ATCC #BAA-2469) and Klebsiella pneumoniae pneumoniae(ATCC #BAA-2146).

Testing was conducted in accordance with a Non-GLP evaluation of onetest material for its antibacterial properties when challenged with twomicroorganism species using an in-vitro Time-Kill Method. The testmaterial was prepared at two different concentrations prior toevaluation. Test Solution #1 was prepared by diluting the test material(Composition A) in the ratio of 1.0 mL of concentrated test material to100 mL of sterile Water-for-Irrigation, USP (WFI) (1:100 [v/v]dilution). Test Solution #2 was prepared by diluting the test materialin the ratio of 0.1 mL of concentrated test material to 100 mL of WFI(1:1,000 [v/v] dilution). A 0.1 mL aliquot of a challenge suspension wasinoculated into a test tube containing 9.9 mL of a test solution andmixed thoroughly using a vortex mixer. Each challenge suspension wasexposed to each Test Solution for 10 minutes, timed using a calibratedminute/second timer. After the exposure time had elapsed, a 1.0 mLaliquot was transferred from the tube containing Test Solution/inoculuminto a separate sterile test tube containing 9.0 mL of Butterfield'sPhosphate Buffer solution with product neutralizers (BBP++), and mixedthoroughly using a vortex mixer. Ten-fold dilutions were prepared inneutralizing solution, mixing thoroughly using a vortex mixer betweendilutions. 1.0 mL and/or 0.1 mL aliquots of each dilution werepour-plated, in duplicate, using Tryptic Soy Agar with productneutralizers (TSA+).

Tables 3 and 4 present the initial population (CFU/mL) and post-exposurepopulations (CFU/mL) of each challenge species, and the Log₁₀ andpercent reductions produced by each Test Solution following a 10-minuteexposure.

TABLE 3 Test Solution #1 - Composition A 1:100 [v/v] dilution²Microorganism Species Inoculum Exposure Post-Exposure Log10 Percent(ATCC #) Level Time Population Reduction Reduction Escherichia coli8.450 × 10⁶ 10 minutes <1.00 × 10¹ 5.9269 99.9999% (ATCC #BAA-2469)Klebsiella pneumoniae 2.350 × 10⁷ 10 minutes <1.00 × 10¹ 6.3711 99.9999%pneumoniae (ATCC #BAA-2146)

TABLE 4 Test Solution #2 - Composition A 1:1,000 [v/v] dilution²Inoculum Post-Exposure Microorganism Species Level Exposure PopulationLogw Percent (ATCC #) (CFU/mL) Time (CFU/mL) Reduction ReductionEscherichia coli 8.450 × 10⁶ 10 minutes <1.00 × 10¹ 5.9269 99.9999%(ATCC #BAA-2469) Klebsiella pneumoniae 2.350 × 10⁷ 10 minutes <1.00 ×10¹ 6.3711 99.9999% pneumoniae (ATCC #BAA-2146)

Notes:

-   -   2. Concentrated Test Product (composition A) was diluted with        sterile Water-for-Irrigation, USP, prior to evaluation.

Example 3 Efficacy Study of Anti-MRSA Composition A on Animal Model

Efficacy study on the composition A was conducted in C57b1-6 Mice andSprague Dawley rats.

3a: Methicillin-Resistant S. aureus (MRSA) Intra-Venous Administrationin Mice (25 Mice C57BL16):

A suitable infectious dose of 2.4×10⁷ CFU/ml was provided by 0.1 mL ofbacterial stock injected intravenously in each mouse. Oral or IPadministration of the composition A occurred 15 minutes after injectionof the bacteria and once daily for a further five days.

Five groups of five C57BL/6 Mice each were used and the groups andtreatment dosage dose were as shown in Table 5:

TABLE 5 Group Dose Dose Number of mice Treatment Dosage Level VolumeAnimals 1 Control na na 400 ul/200 g 5 saline (oral gavage) 2 IP  500mg/kg MTD 10 ul/20 g 10 3 oral gavage 2000 mg/kg MTD 40 ul/20 g 10 na =not applicable

Results

Control Group 1: All 5 mice from the control group died consecutively ondays 5, 6, and 7 from bacterial infections. One day prior to death,there were observations of sickness, including apatia, restriction ofmovement, and loss of appetite. They were found dead overnight.Histological changes could not be observed from the carcasses.

IP Group 2: In the itraperitoneal group, two of ten mice (20%) died onthe third day of the study after the third injection of High Dose (LD50)500 mg/kg Composition A. The cause of death was not confirmed. Theremainder of the mice (80%) survived post day 5 including MRSAadministration with no apparent sickness.

Gavage Group 3: In the gavage group, two of ten mice (20%) died; oneeach on days 3 and 7. The remainder of the mice (80%) survived post day5 including MRSA administration with no apparent sickness.

3b: (MRSA) Intra-Venous (Tail Vein) Administration in Mice (25 MiceC57BL/6)

Mice were inoculated intravenously with 1×10⁷CFU/ml of MRSA. The testcomposition was administered to the inoculated mice by oral gavage. Atthe end of study, post 14 days, blood was collected and cultured forbacteria detection.

Five groups of five C57BL/6 Mice each were used and the groups were asfollows:

-   -   1) 5 mice (G1) control (only MRSA)    -   2) 5 mice (G2) treatment 500 mg/kg of Composition A, once/day        for 14 days IV induction of MRSA    -   3) 5 mice (G3) treatment 100 mg/kg of Composition A, once/day        for 14 days IV induction of MRSA    -   4) 5 mice (G4) treatment 50 mg/kg of Composition A, once/day for        14 days IV induction of MRSA    -   5) 5 mice (G5) treatment 25 mg/kg of Composition A, once/day for        14 days IV induction of MRSA

Results

Blood cell culture results for groups G1 to G5 are shown in FIGS. 1A to1F. With the exception of Group 1 all blood cultures from all dosedtreatment animals did not show any bacterial growth. In the controlgroup G1, mouse no. 5 died on day 10, post IV administration ofStaphylococcus aureus. All the rest of the animals (four animals) showedmassive bacterial growth. The health of the animals in the treatmentgroups was not affected.

4b: Rat Full-Thickness Excision Model on Sprague-Dawley Rats.

Procedure (Summary):

a) Wound sites were prepared on the back of each anesthetized rat byexposing ˜2 cm² of fascia.

b) The exposed fascia was inoculated with a 100-μL suspension of 10⁷CFU/mL of ATCC USA300 S. aureus.

c) After 15 minutes, the wounds were treated with 0.4 ml of CompositionA or with sterile saline (recovery controls) and repeated daily for 5consecutive days. Five days following treatment, a swab was taken fromthe wound and cultured on tryptic soy agar, incubated overnight at 35°C., and colonies were counted to determine organism survival.

d) Three groups of three adult male rats each were used and the groupswere as follows:

Groups:

Total animals: 9 rats received topical or oral (gavage) administrationof test composition or saline control once daily for 5 days.

Group 1) 3 rats (control) received saline (400 uL/200 g rat); topical,1× daily for 5 days

Group 2) 3 rats treated with 2000 mg/kg Composition A (400 uL/200 grat); topical 1× daily/5 days

Group 3) 3 rats treated with 500 mg/kg Composition A (100 uL/200 g rat);oral administration 1× daily/5 days

Group Assignments and Dose Levels

Group Dose Dose Number of rats Treatment Dosage Level Volume Animals 1Rat wound 2000 mg/kg ND 400 uL/200 gr 3 topical rat application 2 Ratwound  500 mg/kg ND 100 uL/200 gr 3 oral rat gavage 3 Rat wound 2000mg/kg na 400 uL/200 gr 3 control (saline) rat ND = not determined; na =not applicable

Results

Control Group

All animals showed evidence for an active bacterial infection in thearea of the wound. A thick yellow/green opaque liquid was produced inthe infected tissue, consisting of dead white blood cells and bacteriawith tissue debris and serum. This was observed in all 3 rats fromcontrol group. All animals survived.

Topical Group

In the topical application group (see images in appendix for Group 2),there was a significant improvement in the appearance of the wound,showing no puss or any kind of secretion compared to the “no treatment”group (saline), where the wound showed infection. Slight necrosis wasobserved post day 5 in “treatment group” (topical application). Thiseffect may be due to the test material (2000 mg/kg treatment withrepeated 5 day administration). No signs of sickness were observe in thetopical application group.

Gavage Group

In the gavage group, all rats died on days 3, 4, and 5. The dose of 500mg/kg may have produced the death of rats by the accumulation of thecomposition in repeated administration.

4b: Rat Full-Thickness Excision Model on Sprague-Dawley Rats.

Wound sites were prepared on the back of each anesthetized rat byexposing ˜2 cm2 of fascia. Five adult male Sprague-Dawley rats, with onewound site each were used for each treatment group. The exposed fasciawas inoculated with a 100-μL suspension of 1×10⁷ CFU/mL of ATCC us, 300S. aureus. After 15 minutes, the wounds were treated, via topicalapplication, with 0.4 ml of Composition A or with sterile saline(recovery controls) and repeated daily for 14 consecutive days. Fourteendays following treatment, a swab taken from the wound was cultured ontryptic soy agar, incubated overnight at 35° C., and colonies werecounted to determine organism survival.

Five groups of five Sprague-Dawley Rats each were used and the groupswere as follows:

-   -   1) 5 Rats (G1) control (only MRSA)    -   2) 5 Rats (G2) treatment 25 mg/kg of Composition A, once/day for        14 days direct wound application induction of MRSA    -   3) 5 Rats (G3) treatment 50 mg/kg of Composition A, once/day for        14 days direct wound application induction of MRSA    -   4) 5 Rats (G4) treatment 100 mg/kg of Composition A, once/day        for 14 days direct wound application induction of MRSA    -   5) 5 Rats (G5) treatment 500 mg/kg of Composition A, once/day        for 14 days direct wound application induction of MRSA

At the end, post 14 days, a swab from wound was collected and culturedfor bacteria detection.

Results

In Group 1, multiple colonies were present in all five blood cellculture agar plates from all five rats. In Group 2, the blood agarculture presents two small colonies from rat no. 7, and rat no. 9. InGroup 3, the blood agar culture plates showed presence of four smallcolonies from rat nos. 12 to 14. In Group 1, zero colonies were observedin the blood agar culture plates.

All groups with treated wounds show no sign of infection. All woundswere 100% healed. In the control group the wound was infected.

Example 5 Oral Dose and Topical Application Toxicity Study

Repeated dose oral toxicity and topical application toxicity study wasconducted in BALB/C mice to ascertain the safety of Composition Afollowing repeated oral or topical administration.

5a: Two Weeks Repeated Dose Oral Toxicity Study

Procedure

Composition A was administered to BALB/C mice (n=5 males and 5 femalesper group) daily by oral gavage at doses of 0 mg/kg/day (40 μl/20 golive oil), 1000 mg/kg/day (40 μl/20 g composition A), 300 mg/kg/day (40μl/20 g composition A), and 100 mg/kg/day (40 μl/20 g composition A) for14 days.

During the dosing period, the animals were observed daily immediatelyafter administration and again 6 hours post administration for clinicalsigns of toxicity.

Animals that died or were terminated in a moribund state during the testperiod were necropsied, the organs weighed, and collected forhistopathological analysis. Surviving animals were terminated 24 hoursafter the necropsied, the organs weighed, and fixed forhistopathological analysis.

Blood and urine was collected and analyzed from each animal at time 0(pre-dose), Day 1 (24 hrs.), Day 7, and Day 14 (end of study) forhematology, CBC, blood chemistry and urinalysis.

Results

Oral administration of the test composition at doses up to 1000mg/kg/day showed no effect on body weight or body weight gain in eithermale or female mice.

Hematology—Some significantly different numbers at any dose are not drugrelated.

Clinical Chemistry—There was no drug effect at any dose

Urine Analysis—There was no drug effect at any dose or time point

Gross Pathology—We concluded that the observed differences were notrelated to the test composition

Histopathology—

Some findings or incidental findings were not related to composition Aat any doses.

Throughout the study duration of 14 days, no sickness was observed inthe mice.

The above results confirmed that oral administration of Composition A ina high dose (1000 mg/kg) did not produce any adverse effect.

5b: Two Weeks Repeated Dose Topical Application Toxicity Study

Procedure

Composition A was administered to BALB/c mice (n=5 males and 5 femalesper group) daily by topical application at doses of 0 mg/kg/day (40μL/20 g olive oil), 1000 mg/kg/day (40 μL/20 g composition A), 300mg/kg/day (40 μL/20 g composition A), and 100 mg/kg/day (40 μL/20 gcomposition A) for 14 days.

Each mouse was placed under isofluorane gas anesthesia; on the back ofthe shoulder, the fur was clipped and a 3-4 mm circular section of skinwas removed creating an artificial wound. The composition was applieddaily for 14 days over the created wound. The wound area was measured onday 0, day 7, and day 14.

During the dosing period, the animals were observed daily immediatelyafter administration and again 6 hours post administration for clinicalsigns of toxicity.

Animals that died or were terminated in a moribund state during the testperiod were necropsied, the organs weighed, and collected forhistopathological analysis. Surviving animals were terminated 24 hoursafter the last dose, necropsied, the organs weighed, and fixed forhistopathological analysis.

Blood and urine was collected and analyzed from each animal at time 0(pre-dose), Day 1 (24 hrs.), Day 7, and Day 14 (end of study) forhematology, CBC, blood chemistry and urinalysis.

Results

Topical administration of the test composition at doses up to (1000mg/kg) showed no effect on body weight or body weight gain in both maleand female mice.

Hematology—Some significantly different numbers at any dose are not drugrelated.

Clinical Chemistry—There was no drug effect at any dose.

Urine Analysis—There was no drug effect at any dose or time point.

Gross Pathology—We concluded that the observed differences were notrelated to the test composition.

Histopathology—Some findings or incidental findings were not related tocomposition A at any doses.

Throughout the study duration of 14 days, no sickness in the mice wasobserved

The above results confirmed that topical application of Composition A ina high dose (1000 mg/kg) did not produce any adverse effect.

Example 6 Toxicokinetics Study for Oral Dose and Topical ApplicationToxicity

Toxicokinetics study for two weeks repeat dose oral toxicity and topicalapplication toxicity of anti-bacterial Composition A was conducted inBALB/C mice to determine the amount/level of undecylenic acid analytefrom Composition A in K2EDTA plasma samples in mice at different timepoints following the administration.

An LC/MS/MS procedure (M150911) was developed for the quantification ofundecylenic acid in mouse K2EDTA plasma. Undecylenic acid and theinternal standard (9-Decenoic acid) were isolated from mouse K2EDTAplasma by liquid-liquid extraction (M1BE was used as solvent). Theextracted samples were transferred to clean injection vials. A 5 μLsample was injected into the LC/MS/MS system for analysis. The standardcurve range was 0.5-100 μg/mL of undecylenic acid in K2EDTA plasma. A 50μL K2EDTA plasma sample aliquot was used for sample preparation andanalysis. All study samples were within the stability parametersestablished during validation of the method. The stability parametersinclude reinjection stability of extracted samples for up to 29.8 hourson autosampler at 15° C.; refrigeration stability of extracted samplesfor up to 69.4 hours at 2-8° C.; bench-top stability of unextractedsamples for up to 5.5 hours; and freeze-thaw stability for up to fourfreeze-thaw cycles, and long-term storage stability of the QC samplesfor up to 25 days at −70° C. (long enough to cover the study samplestorage period).

Each analytical batch contained one set of calibration standards placedat the beginning of the run. The peak areas for undecylenic acid and theinternal standard were determined using the Analyst software. Aquadratic regression (weighted 1/×2) was applied to a plot of the peakarea ratio versus concentration for the standards to obtain thecalibration curve. The sample concentrations are calculated from thecurve parameters as performed by the Analyst software version 1.4.2.

6a: Toxicokinetics for Oral Dose

Procedure

Composition A was administered to BALB/C mice (n=18 males and 18 femalesper group) by oral gavage at doses of 0 mg/kg/day (40 μl olive oil),1000 mg/kg/day (40 μl composition A), 300 mg/kg/day (40 μl compositionA), and 100 mg/kg/day (40 μl composition A) and serial blood sampleswere taken from six mice per time point (3, 6, 8, 12, and 24 hours) onDay 1 and Day 14 of administration.

-   -   1. Group 1 Control—time 0. Blood samples were collected from six        mice (3 males and 3 females).    -   2. Group 2 High Dose—1000 mg/kg—time 3, 6, 8, 12, and 24 hours        on Day 1 and Day 14 of administration. Blood samples from six        mice (3 males and 3 females) were collected at each time point.    -   3. Group 3 Medium Dose—300 mg/kg—time 3, 6, 8, 12, and 24 hours        on Day 1 and Day 14 of administration. Blood samples from six        mice (3 males and 3 females) were collected at each time point.    -   4. Group 4 Low Dose—100 mg/kg—time 3, 6, 8, 12, and 24 hours on        Day 1 and Day 14 of administration. Blood samples from six mice        (3 males and 3 females) were collected at each time point.

Blood was collected by cardiac puncture (approx. 500 μL), centrifuged,the plasma collected and pooled at each time point, then frozen at −80 Cfor future analysis.

Results

Oral administration of the test composition at a dose of 100 mg/kgresulted in no detectable analyte in the plasma of male mice 3 hoursafter administration. In female mice, one animal showed detectablelevels of the test composition at this same dose level.

Oral administration of the test composition at a dose of 300 mg/kgresulted in detectable levels in the serum of both male and female micethree hours after administration. Serum plasma analyte concentrations atthe 3 hour time point was less than 3 times higher than the serumanalyte level seen in the one female mouse at the low dose (1.56 vs 2.35ug/mL). By six hours after administration, only two of three males orfemales had detectable serum analyte levels while at 8 hours post dose,no males and one female had detectable analyte plasma levels.Interestingly, one male also showed detectable analyte levels at 24hours at levels similar to that seen at 6 hours, however, serum samplescollected at 8 and 12 hours showed no detectable analyte levels.

Oral administration of the test composition at the highest dose level(1000 mg/kg/day) resulted in detectable analyte levels in the serum intwo of three males and all three females. Analyte levels between thesefive animals were variable ranging from 0.66 to 6.57 ug/ml. However, thehighest value observed, 6.57 ug/mL was approximately 3 times greaterthan the highest serum analyte level seen at the 300 mg/kg/day doselevel sample. Sequential serum samples with detectable analyte werepresent in two of the six animals tested. Of the six animals tested,only one had three sequential serum samples with measurable analytelevels. Consequently, no AUC determinations could be performed. Inaddition, due to the limited number of animals and the variable natureof the values obtained, Cmax values are also not reliably determinable.

6b: Toxicokinetics for Topical Application

Procedure

Composition A was administered to BALB/C mice (n=18 males and 18 femalesper group) by topical application at doses of 0 mg/kg/day (40 μl oliveoil), 1000 mg/kg/day (40 μl composition A), 300 mg/kg/day (40 μlcomposition A), and 100 mg/kg/day (40 μl composition A) and serial bloodsamples were taken from six mice per time point (3, 6, 8, 12, and 24hours) on Day 1 and Day 14 of administration.

-   -   1. Group 1 Control—time 0. Blood samples were collected from six        mice (3 males and 3 females).    -   2. Group 2 High Dose—1000 mg/kg—time 3, 6, 8, 12, and 24 hours        on Day 1 and Day 14 of administration. Blood samples from six        mice (3 males and 3 females) were collected at each time point.    -   3. Group 3 Medium Dose—300 mg/kg—time 3, 6, 8, 12, and 24 hours        on Day 1 and Day 14 of administration. Blood samples from six        mice (3 males and 3 females) were collected at each time point.    -   4. Group 4 Low Dose—100 mg/kg—time 3, 6, 8, 12, and 24 hours on        Day 1 and Day 14 of administration. Blood samples from six mice        (3 males and 3 females) were collected at each time point. Blood        was collected by cardiac puncture (approx. 500 μL), centrifuged,        the plasma collected and pooled at each time point, then frozen        at −80 C for future analysis.

Results

Application of test composition topically at a dose of 100 ug/kg/dayresulted in no detectable serum analyte levels in either male or femalemice. At the 300 mg/kg/day dose level, serum analyte levels were belowthe level of detection in the male mice. In the female mice, at the 3hour time point, two animals had detectable serum analyte levels. Asthese values were approximately half of what was observed in female miceorally administered the composition, it is possible that the serumanalyte levels were secondary to grooming. Further support for thishypothesis comes from the one male animal with detectable serum analytelevels 3 hours after topical administration. In this case, the mouse hadserum analyte levels that were comparable to that seen with oraladministration. Based on these data, it is possible that the testcomposition is absorbed through the skin but it is equally likely thatthe serum analyte levels seen following topical administration at themiddle dose are the result of the animals grooming.

At the 1000 mg/kg/day dose level, two males and two females haddetectable serum analyte levels 3 hours post application. The serumanalyte levels were lower than that seen following topicaladministration at the 300 mg/kg/day dose providing further support fororal uptake secondary to grooming.

The results from toxicokinetics studies suggest that absorption eitherby the oral or topical route appears to be limited. As a result,systemic exposures are also limited.

Example 7 Comparative Efficacy Study

Efficacy Study in comparison to leading anti-MRSA antibiotics wasconducted in BALB/C mice.

7a. Administration Route: Oral Gavage

1) vancomycin

Group 1 (6 mice males)—MRSA Induction IP no treatment

Group 2 (6 mice males)—MRSA Induction IP—oral treatment for 7 days

Group 3 (6 mice males)—No Induction—oral treatment for 7 days

Group 4 (6 mice males)—MRSA Induction IP—oral treatment with compositionA

2) clindamycine

Group 1 (6 mice males)—MRSA Induction IP no treatment

Group 2 (6 mice males)—MRSA Induction IP—oral treatment for 7 days

Group 3 (6 mice males)—No Induction—oral treatment for 7 days

Group 4 (6 mice males)—MRSA INDUCTION IP—oral treatment with compositionA

3) erythromycin

Group 1 (6 mice males)—MRSA Induction IP no treatment

Group 2 (6 mice males)—MRSA Induction IP—oral treatment for 7 days

Group 3 (6 mice males)—No Induction—oral treatment for 7 days

Group 4 (6 mice males)—MRSA Induction IP—oral treatment with compositionA

7b. Administration Route: Topical Application

1) Bactroban

Group 1 (6 mice males)—MRSA Induction (wound application) no treatment

Group 2 (6 mice males)—MRSA Induction (wound application)—treatment for7 days

Group 3 (6 mice males)—No Induction—topical application treatment for 7days

Group 4 (6 mice males)—MRSA Induction (wound application)—treatment withcomposition A

Dosage for Comparative Test

For Composition A Dose was 100 mg/kg. For the antibiotics the dose wasas per RX prescription converted to mice Body Weights.

1—Erythromycin 250 mg/pt b.i.d. equals 2.1 mg/20 g mouse/day 300 mgtotal/12 mice

Erythromycin 500 mg/pt b.i.d. equals 4.2 mg/20 g mouse/day

2—Clindamycin 150 mg/pt b.i.d. equals 1.25 mg/20 g mouse/day 200 mgtotal/12 mice

Clincamycin 300 mg/pt b.i.d. equals 2.5 mg/20 g mouse/day

3—Vancomycine 250 mg/pt b.i.d. equals 2.1 mg/20 g mouse/day 300 mgtotal/12 mice

4—A small amount (0.1 g) of Bactroban Ointment was applied to theaffected area /20g mice/day.

Results

Blood cell culture results for the comparative tests are as discussedbelow:

1. Vancomycin Group

Group 1—MRSA induction—No treatment: There was bacteria development fromthe blood taken from mice no. 1, 4, 5, 6.

Group 2—MRSA induction vancomycin treatment: There was bacterial growthone colony from animal no. 4 blood culture.

Group 3—NO MRSA induction vancomycin treatment; all probes negative.

Group 4—MRSA induction Composition A treatment: Negative.

The comparison between the treatments shows a slight advantage for theComposition A treatment, where no bacteria was found versus thevancomycin treatment that shows one colony.

2. Erythromycin Group

Group 1—MRSA induction No treatment: All six animals have positivecolonies.

Group 2—MRSA induction; Erythromycin treatment: show two colonies inmice nr.1 and nr.2.

Group 3—No MRSA treatment with erythromycin: all probes negative but thepictures show some light reflection between probes 3 and 4, and, 5 and6.

Group 4—MRSA induction with treatment with composition A: All probesnegative—pictures show some light reflections.

The comparison between the treatment show a definite superior results inthe groups treated with Composition A (negative results) anderythromycin that presents 2 positive colonies.

3. Clindamycin Group

Group 1—MRSA induction—No treatment: All samples show positive colonies.

Group 2—MRSA induction—Clindamycin treatment: All samples show negativecolonies (light reflection effect at probe 1, 5 and 6).

Group 3—No MRSA—Clindamycin treatment: All negative (light effects atsamples 1, 2, 3 and 6).

Group 4—MRSA induction Treatment with Composition A: All samplesnegative, light effects at no. 6 and no. 2.

The comparison between the treatment with Clindamycin and Composition Ashow both a positive effect.

4. Bactroban Group—Topical Application

Group 1—MRSA induction—no treatment: all samples show positive colonies.

Group 2—MRSA induction—Bactroban treatment: Blood probes negative, woundswab positive in mouse nr. 4, (5 colonies).

Group 3—no MRSA induction—Bactroban treatment: all negative.

Group 4—MRSA induction—Composition A treatment: blood probes negative,one probe positive from swab in animal nr. 2, (1 colony).

Comparison between Bactroban and Composition A topical application showa slight advantage for Composition A (five colonies in Bactroban and onecolony in Composition A).

In addition Composition A presents the advantage that it spreads anddiffuses all over the infected wound areas without touching thecontaminated area, and can be applied by droplets versus Bactroban thatneeds to be applied in direct contact with the wound on the contaminatedarea. The Composition A used had the lowest concentration of 100 mg/kg.

Example 7 OECD Bovine Corneal Opacity and Permeability Test (BCOP)

Composition A was tested for potential ocular irritation using analternative to the Draize methodology. This protocol is based on themethodology described in the current OECD Guideline for the Testing ofChemicals #437.

Method:

Three bovine corneas per group were dosed with 0.75 ml of composition A,Minimal Essential Media (MEM) (negative control), or 100% Ethanol(positive control). Following a 10-minute exposure for each group ofdosed corneas, opacity measurements and sodium fluorescein permeabilitywere determined. The results are summarized in Table 6:

TABLE 6 Corrected In Vitro Irritation Corrected Mean Mean TreatmentScore (IVIS) Opacity Score Optical Density composition A 0.50 0.33 0.011MEM 0.76 0.67 0.006* (negative control) 100% Ethanol 32.34 22.66 0.645(positive control)

Based on an In Vitro Irritation Score of less than 3, in accordance withEURL DB-ALM protocol No. 127, Composition A was considered to benon-irritant.

Example 8 3T3 Neutral Red Uptake Phototoxicity Assay

The cytotoxicity and phototoxicity of the test composition to 3T3 cells(in the presence or absence of UVA light) was assessed by Neutral RedUptake. The 3T3 Neutral Red Uptake Phototoxicity Assay (3T3 NRU PT),based on the OECD Guideline for Testing of Chemicals: No. 432, wasdesigned to detect the phototoxicity induced by the combined action of atest composition and solar-simulated UVA+visible light in an in vitrocytotoxicity assay using the BALB/C 3T3 mouse fibroblast cell line asthe test system.

The assay identifies aqueous-soluble compounds (or formulations) thathave the potential to exhibit in vivo phototoxicity after systemicapplication.

An Ultraviolet-Visible light (UV-VIS) spectral scan was performed on asolution containing 0.1% of the test composition in a solution of 1%DMSO/HBSS. The scan showed that most of the absorbance occurred belowthe Ultraviolet A (UVA) and Ultraviolet B (UVB) regions (OD280 to OD400)and should not have any impact on the results of the 3T3 assay. For boththe range-finding screen and the definitive test of the 3T3 assay,BALB/C 3T3 cells were seeded in the central 60 wells of duplicate96-well microplates and maintained in culture for approximately 24hours. The two 96-well plates were then preincubated with eightdifferent concentrations of the test composition for approximately onehour. After preincubation, one plate was irradiated with a dose of 5J/cm² Solar Simulated Light (SSL, containing wavelengths in the UVA andvisible regions with >99% of UVB blocked out), while the duplicate platewas kept in the dark (No SSL). After UV irradiation, the treatmentmedium was replaced with culture medium and, after approximately 24hours, cell viability was determined by neutral red uptake for threehours.

A range finding screen was performed to determine the acceptableconcentrations for the definitive test. MS Excel® was used to calculatethe EC50 values and Photo-Irritant Factor (PIF) for the test article andthe Chlorpromazine (CPZ) positive control in both the Screen and theDefinitive test. Results of the definitive test are summarized in Table6:

TABLE 6 Test Concentration EC50 EC50 + Composition Range Tested No SSLSSL PIF composition 0.0068-0.1% >0.1% >0.1% None A CPZ No SSL: 6.81-100μg/ml + 18.8 μg/ml 0.4 μg/ml 47.0 Positive SSL: 0.22-3.16 μg/ml Control

Test Composition A had EC50 values of >0.1% for both No SSL and +SSL;therefore, the Photo-Irritancy Factor (PIF) could not be calculated.This test composition therefore is not considered to have phototoxicpotential in the 3T3 Neutral Red Uptake Phototoxicity Test.

Example 9 MatTek EpiDerm™ Skin Irritation Test (SIT)

This test was conducted to predict dermal irritation potential of testarticles in the context of identification and classification of skinirritation hazard according to the European Union (EU) classification(R38 or no label), United Nations Globally Harmonized System ofClassification and Labeling of Chemicals (GHS) classification system(Category 2 and non-irritants), and OECD Guideline for the Testing ofChemicals No. 439—In Vitro Skin Irritation: Reconstructed HumanEpidermis Test Method. This study was designed based on MatTek protocolin vitro EpiDerm™ Skin Irritation Test.

MatTek EpiDerm™ tissue samples were treated in triplicate with the testcomposition, Negative Control and Positive Control for 60 minutes.Following treatment and subsequent incubation time, the viability of thetissues was determined using Methyl thiazole tetrazolium (MTT) uptakeand reduction. The absorbance of each sample was measured at 540 nm. Theviability was then expressed as a percent of control values. If the meantissue viability was ≦50%, the test material was classified as anirritant; if the mean tissue viability was ≦50%, the test material wasclassified as a non-irritant. The results are summarized in table 7:

TABLE 7 Test and Control Article Irritancy Identity Mean TissueViability Classification Composition A 106.7% Non-Irritant PhosphateBuffered Saline 100.0% Non-Irritant (Negative Control) 5% Sodium DodecylSulfate 3.1% Irritant (Positive Control)

Example 10 Preservative Challenge Test

The USP-NF <51>—Preservative Challenge Test was conducted to evaluatethe antimicrobial activity of the test composition for use in cosmeticsand personal care products.

TABLE 8 Results of Challenge tests analysis 10 minutes after 7 daysMicro-organism Initial inoculums innoculation after Staphylococcus 1.6 ×10⁴ <10 aureus (cfu/g) Staphylococcus 7.3 × 10⁵ <10 <10 aureus (cfu/g)E. coli (cfu/g) 4.4 × 105 <10 <10 Pseudomonas 3.5 × 105 <10 <10aeruginosa (cfu/g) Candida albicans 3.6 × 105 <10 <10 (cfu/g)Aspergillus niger 6.2 × 105 <10 <10 (cfu/g)

Example 11 Evaluation Antimicrobial Activity of a Known Tegaderm andEffect of Composition A

The cellulose part of Tegaderm 3584 from 3M was tested for antimicrobialactivity, which did not show any antimicrobial activity against variousbacterial species. Introduction of 5 mg of composition A to 3M cellulosefibre (500 mg) of Tegaderm, resulted in elimination of 99.99% of widerange of bacteria including E. coli.

Example 12 Evaluation of Antimicrobial Activity of Composition AIncorporated Paper Product via Dilution Test

a) 200 microliter of E. coli B was grown in 10 ml of TSB mediumovernight. Serial dilution test showed high E. coli activity (108CFU/mL) in petri dish. 200 μL of E. coli B and 200 μL of composition Awere dispersed in 10 ml of TSB medium overnight. Serial dilution testshowed no E. coli activity, concluding that incorporation of compositionA resulted in 99.999% E. coli B and bacteriophage resistance E. coli B.

b) 0.4 g spruce/pine/fir (SPF) pulp paper sheet patch containing 200 μLof composition A was immersed in 10 mL TSB medium containing 200 μL E.coli. Serial dilution test showed no E. Coli activity.

c) 0.4 g SPF paper sheet patch containing 200 μL of composition A wasimmersed in 10 mL overnight cultured E. coli (fully grown 108 CFU/mL).Serial dilution test showed four log of reduction in E. coli activity(correspond to 80% drop in E. coli activity.

Example 13 Evaluation of Antimicrobial Activity of Composition AIncorporated Plastic Product via Serial Dilution Test

1.0 g of polyethylene (PE, melting 90˜100° C., Mw of 4000) was heated tomelt (110° C.), then 500 μl of Composition A was added to PE meltsolution. The mixture was placed in flat petri dish to reach roomtemperature and solidified to film. About 0.5 g of sample(PE-composition A) was immersed in E. coli suspension overnight at 37°C. Complete E. coli removal was observed after serial dilution test (108CFU/mL to Zero).

DSC analysis showed that composition A and PE are not miscible blend andas a result a complete diffusion release of composition A was confirmed.Physical structure property of PE should remain same (further DMAanalysis is required to confirm it.)

SEM analysis of PE/composition A showed that composition A is uniformlyembedded through PE film

Same results were obtained when composition A was mixed with PE havingMW of about 125 k and 100K (complete removal of E. coli).

1. An antibacterial composition comprising: a) about 40% to about 80% ofan unsaturated fatty acid or a pharmaceutically acceptable salt thereof,by weight of the total weight of the composition, wherein theunsaturated fatty acid is selected from a unsaturated fatty acid havingfrom 8 to 12 carbon atoms; b) about 10% to about 40% of lactic acid or apharmaceutically acceptable salt thereof, by weight of the total weightof the composition; about 5% to about 25% of at least one amino alcohol,by weight of the total weight of the composition, wherein the aminoalcohol has a formula:

wherein R¹ and R³ are each independently methyl, R² and R⁴ are eachmethyl, or R² is methyl and R⁴ is CH₂OH; wherein the composition is freeof additional antimicrobial or biocidal agents.
 2. The antibacterialcomposition according to claim 1, comprising: about 50% to about 70% ofsaid unsaturated fatty acid; about 20% to about 30% of lactic acid; andabout 10% to about 15% of said amino alcohol.
 3. The antibacterialcomposition according to claim 1, comprising; about 55% to about 65% ofsaid unsaturated fatty acid; about 25% to about 35% of lactic acid; andabout 10% to about 20% of 95% of said amino alcohol.
 4. Theantibacterial composition according to claim 1, wherein the fatty acidis undecylenic acid or a pharmaceutically acceptable salt thereof. 5.The antibacterial composition according to claim 1, wherein the aminoalcohol is 2-amino-2-methyl-1-propanol.
 6. The antibacterial compositionaccording to claim 1, comprising: about 50% to about 60% of undecylenicacid; about 25% to about 30% of lactic acid; and about 10% to about 20%of 95% AMP.
 7. A pharmaceutical formulation comprising a composition asdefined in claim 1, and a pharmaceutically acceptable carrier.
 8. Thepharmaceutical formulation according to claim 7, where said formulationis for topical administration.
 9. The pharmaceutical formulationaccording to claim 8, where said formulation is in the form of a cream,lotion or a gel.
 10. The pharmaceutical formulation according to claim7, where said formulation is for oral administration.
 11. Thepharmaceutical formulation according to claim 7 for inhibiting growthand/or proliferation of bacteria.
 12. A method of killing and/orinhibiting the growth of microbes on a substrate comprising applying aneffective amount of the antibacterial composition as defined in claim 1to the substrate.
 13. (canceled)
 14. A cosmetic product, personal careproduct, cleanser, polish, paint, spray, soap, detergent, paper productor plastic product comprising the composition as defined in claim
 1. 15.A method of treatment or prevention of a bacterial infection, or adisease or disorder associated therewith, in an subject in need thereof,comprising administering an effective amount of the antibacterial asdefined in claim
 1. 16. The method of claim 15, wherein said compositionis used in combination with one or more anti-microbial agent(s).
 17. Atransdermal patch comprising the composition as defined in claim
 1. 18.An antibacterial composition consisting essentially of: a) about 40% toabout 80% of an unsaturated fatty acid or a pharmaceutically, acceptablesalt thereof, by weight of the total weight of the composition, whereinthe unsaturated fatty acid is selected from a unsaturated fatty acidhaving from 8 to 12 carbon atoms; b) about 10% to about 40% of lacticacid or a pharmaceutically acceptable salt thereof, by weight of thetotal weight of the composition; c) about 5% to about 25% of at leastone amino alcohol, by weight of the total weight of the composition,wherein the amino alcohol has a formula:

wherein R¹ and R³ are each independently H or methyl, R² and R⁴ are eachmethyl, or R² is methyl and R⁴ is CH₂OH.